Method for diagnosing tumors using mouse monoclonal antibodies

ABSTRACT

The subject invention relates to monoclonal antibodies and uses thereof. In particular, the invention relates to three monoclonal antibodies, referred to as B1, B3, and B5, which are useful in the treatment and diagnosis of many forms of cancer.

This is a Division of application Ser. No. 08/051,133, filed Apr. 22,1993, now abandoned, which is a Division of application Ser. No.07/596,289 filed Oct. 12, 1990, now U.S. Pat. No. 5,242,813.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to monoclonal antibodies and uses thereof.

In particular, the invention relates to three monoclonal antibodies,referred to as B1, B3 and B5, which are useful in the treatment anddiagnosis of many forms of cancer.

2. Background Information

Current therapies for metastic human cancers, such as radiation orchemotherapy, center on agents that selectively kill rapidly growingcancer cells. Unfortunately, many tumors do not show an unusually fastgrowth rate compared to important normal tissues, such as bone marrow orthe epithelium of the gastrointestinal tract. An alternative group oftherapeutic approaches targets unique chemical structures on the surfaceof tumor cells for therapy, most often employing antibodies that bindselectively to these target molecules. One of these therapeuticapproaches employs antibodies that are coupled to cell-killing agents,such as plant or bacterial toxins. These antibody-toxin complexes,immunotoxins, have been shown to be capable of selectively killing tumorcells in model tumor systems in tissue culture and in laboratory animals(Pastan, et al, Cell, 47:641-48 (1986)). In spite of many attempts toisolate such tumor-specific antibodies for human therapy, there arestill very few antibodies identified that selectively bind only to tumorcells and not to other important normal tissues. Isolation of suchtumor-specific antibodies is, therefore, of importance for theapplication of such immuno-directed therapies.

Monoclonal antibody methodology as originally described by Kohler andMilstein (Nature 156:495-97 (1975)) and disclosed in Koprowski, et al.(U.S. Pat. No. 4,172,124) has allowed the isolation of antibodies inpure form for the construction of therapeutic agents. However, twoproblems have prevented the application of many previously isolatedantibodies. First, many monoclonal antibodies reactive with tumor cellsalso react with important normal human tissues. Secondly, many of theisolated antibodies bind to surface elements that do not efficientlymediate the entry of toxin conjugates into cells by endocytosis. Thepresent invention includes three monoclonal antibodies, B1, B3, and B5,that selectively bind to some human tumors, but not to many importantnormal tissues. These antibodies, when incorporated as the targetingelement of an immunotoxin, also have been shown to allow efficient entryof these toxic agents into cells.

Previously, antibodies reactive with the Lewis Y antigen have beenisolated and characterized. Recently, two antibodies, BR64 and BR96 havebeen described (Hellstrom et al., Cancer Res., 50:2183-90 (1990)) thatreact with Lewis Y antigen, one of which (BR64) is not useful forimmunotherapy because of its reactivity to capillaries in human cardiacmuscle. BR96, however, shows reactivities that might make an immunotoxinconstructed with this antibody potentially useful. The three newmonoclonal antibodies, B1, B3, and B5, referred to above, which wereisolated using a different cell type for immunizations and usingmorphologic screening methods, are similar, but not identical, to BR96.These differences in reactivity to tumors, normal tissues, andcarbohydrate epitopes make these three new antibodies potentially usefulfor the therapy and diagnosis of some forms of human cancer.

SUMMARY OF THE INVENTION

The subject invention relates to three monoclonal antibodies, referredto as B1, B3 and B5, and to uses thereof.

B1, B3 and B5 exhibit a strong reactivity toward variousmucin-producing, as well as nonmucin-producing primate carcinomas. Thus,these antibodies will be useful in the design of targeted therapeuticagents utilized in the diagnosis and treatment of human cancers.

In particular, the present invention relates to a hybridoma whichproduces a monoclonal antibody specific for a cell surface epitopewherein said epitope is characterized by expression on normal primatetissue, malignant human cultured cell lines and human tumors.

The present invention also includes a monoclonal antibody specific forthe cell surface epitope having the above properties. The class of saidmonoclonal antibody is IgG or IgM.

The malignant human cultured cell lines, referred to above, are selectedfrom the group consisting of A431, MCF-7, HTB 20, and HTB 33. The normalprimate tissue is derived from, for example, the esophagus, bladder orstomach. The human tumor noted above is derived from colon, gastric orovarian carcinomas.

The present invention also relates to three separate hybridomas havingthe accession numbers ATCC HB10569, HB10572, and HB10573, respectively.

The monoclonal antibody produced by the hybridoma of accession numberATCC HB10572 is B1. The monoclonal antibody produced by the hybridoma ofaccession number ATCC HB10573 is B3, and the monoclonal antibodyproduced by the hybridoma of accession number ATCC HB10569 is B5.

Furthermore, the present invention also includes a method of treatingcancer comprising administering to a patient, in need of said treatment,an amount of a conjugate of the monoclonal antibody sufficient to effectsaid treatment. The monoclonal antibody may be conjugated with, forexample, a toxin, radionuclide or chemotherapeutic drug. The toxin maybe, for instance, Pseudomonas exotoxin. The chemotherapeutic drug maybe, for example, vinblastin or daunomycin.

The present invention also includes a method of diagnosing cancer in apatient comprising the steps of:

drawing a blood sample from said patient;

adding a monoclonal antibody to said sample in an amount sufficient toreact with cancer shed antigen to form an antigen-antibody complex; and

detecting whether cancer is present in said patient by measuring thepresence or absence of said complex.

Furthermore, the present invention also includes a method of diagnosingcancer in a patient comprising the steps of

removing a tissue or fluid sample from said patient;

adding the monoclonal antibody to the sample; and

visualizing the presence of the antibody in the sample.

The present invention also includes a pharmaceutical compositioncomprising the monoclonal antibody in a concentration sufficient toinhibit tumor growth, together with a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the antitumor activity of BE-PE^(Arg) 57 in mice. Nudemice (20g) were injected with 3×106 cells subcutaneously on day 0.Treatment with 0.75 ug per dose was given I.P. on days 4, 6 and 8.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In order to produce the B1 and B3 monoclonal antibodies of the presentinvention, mice can be tolerized to normal human kidney membranes andimmunized with MCF-7 cells (May et al., American Type Culture CollectionCatalog of Cell Lines and Hybridomas, (May et al., ATCC 1988) 6th Ed.(1989), Matthew et al., J. Immunol. Methods 100:73-82 (1987) andWillingham et al., Proc. Natl. Acad. Sci. USA 84:2474-78 (1987)). Incontrast, in order to produce the B5 monoclonal antibodies, mice are nottolerized and can be immunized with A431 cells (May et al., supra).Spleens from the immunized mice are then removed, and the suspendedcells can be fused, for example, with AG8 mouse myeloma cells, usingpolyethylene glycol. Appropriate clones can be selected after screeningprocedures have been carried out. One screening procedure may involveselecting clones which react with human colon and gastric cancers andnot with normal human liver, kidney or colon tissues. This selectionprocess is important for isolating clones that react with tumors, ratherthan normal tissue, for the use of such antibodies in selective humanimmunotherapy of cancer.

After subcloning of such antibodies, the isotope of the clones can bedetermined. The present inventors have established that the isotope forthe B1 and B3 clones is IgG_(1k), whereas the isotope for the B5 cloneis IgM. Antibody can be purified from the supernatant of the clones.

Once the antibodies are produced, their properties may then becharacterized. For example, one may characterize precisely which primatetissue epitopes are reactive with the B1, B3 and B5 antibodies.

Reactivity is defined as detectable binding to the surface of livingcells using immunohistochemical methods. Such a determination isnecessary so that target agents may be designed which are toxic totumors but not to important normal tissues.

The distribution of reactivity in normal human tissues, human tumors andnormal cynomolgus monkey tissues is summarized in Table I below.

                                      TABLE I                                     __________________________________________________________________________    Immunohistochemical Localization of B1, B3, B5, and BR96 in Normal Human      and Monkey Tissues                                                                   B1            B3            B5            BR96                         __________________________________________________________________________    NORMAL HUMAN TISSUES                                                          Liver  (-)(5/5)      (-)(5/5)      (-)(1/1)      (-)(1/1)                            (+ large bile duct epith)                                                                   (+ large bile duct)(2/2)    (+ large bile                                                                 duct)(1/1)                   Kidney (-)(5/5)      (-)(5/5)      (+/- apic. tub)(1/2)                                                                        (-)(3/4)(+ ap. d.                                                             tub)(1/4)                    Cardiac                                                                              (-)(6/6)      (10/10)       (-)(6/6)      (-)(6/6)                     Muscle                                                                        Lung   (++ Type II pneum.)(1/2)                                                                    (+ Type II pneum.)(5/7)                                                                     (++ Type II pneum.)(1/1)                                                                    (-)(1/1)                     Cerebral                                                                             (-)(2/2)      (-)(2/2)      (-)(3/3)      (-)(2/2)                     Cortex                                                                        Cerebellum                                                                           (-)(2/2)      (-)(2/2)      (-)(3/3)      (-)(2/2)                     Spinal Cord                                                                          (-)(2/2)      (-)(2/2)      (-)(2/2)      (-)(2/2)                     Pituitary                                                                            (-)(1/1)      (-)(1/1)      (-)(1/1)      nd                           Bone   (-)(2/2)      (-)(1/1)      (-)(1/1)      (-)(1/1)                     Marrow                                                                        Adrenal                                                                              (-)(1/1)      (-)(1/1)      (-)(1/1)      (-)(3/3)                     Spleen (-)(1/1)      (-)(1/1)      nd            (-)(1/1)                     Lymph Node                                                                           (-)(1/1)      (-)(1/1)      (-)(1/1)      nd                           Skin   (-)(1/1)      (-)(2/2)      (-)(2/2)      (-)(3/3)                     Skeletal                                                                             (-)(1/1)      (-)(1/1)      (-)(1/1)      (-)(2/2)                     Muscle                                                                        Peripheral                                                                           (-), ? + cap. endoth.(1/1)                                                                  (-)(? + cap. endoth)(1/2)                                                                   (-)(2/2)      (? + cap. endoth.)(1/1)      Nerve                                                                         Tonsil (+++ epith.)(2/2)                                                                           (+++ epith.)(2/2)                                                                           (+++ epith.)(2/2)                                                                           nd                           Esophagus                                                                            (+++ diff. epith.)(2/2)                                                                     (+++ diff. epith.)(2/2)                                                                     (+++ diff. epith.)(1/1)                                                                     (+++ diff. epith.)(4/4)      Small Bowel                                                                          (+ mucin)(4/4)                                                                              (+ mucin)(3/3)                                                                              (+++ ap muc. gran.)(3/3)                                                                    (+++ muc.)(3/3)              Stomach                                                                              (++++ glnds, muc.)(3/3)                                                                     (++++ glnds, muc.)(3/3)                                                                     (+++ glnds, occ het)(3/3)                                                                   (++++ glnds, muc.)(4/4)      Normal (-)(7/7)      (weak het. epith)(6/6)                                                                      (++ het. muc.)(2/2)                                                                         (-; 3/4)(het. +; 1/4)        Colon                                                                         Bladder                                                                              (+++ epith.)(3/3)                                                                           (+++ epith.)(3/3)                                                                           (+++ epith.)(3/3)                                                                           (+++ epith.)(3/3)            Pancreas                                                                             (#1 = het. + ac.; ducts;)                                                                   (+++ het, d & ac.)(2/2)                                                                     (+++ d & ac.)(1/1)                                                                          (+++ het ac. &                                                                muc.)(2/2)                          (#2 = + acini & ducts)                                                 Salivary                                                                             (+ acini & ducts)(1/1)                                                                      (+++ d & ac.)(1/1)                                                                          (+++ ac. & d.)(1/1)                                                                         (+++ ac. & d.)(2/2)          Gland                                                                         Mammary                                                                              (-)(1/1)      (het. + ducts)(1/1)                                                                         (+++ d, het glnds)(2/2)                                                                     (het. + ducts)(1/1)          Glands                                                                        Epididymis                                                                           (-)(1/1)      (-)(1/1)      (-)(1/1)      nd                           Thyroid                                                                              (-)(1/1)      (+++ colloid, - epith)                                                                      (-)(1/1)      (-)(1/1)                                          (1/1)                                                    Parathyroid                                                                          (-)(1/1)      (-)(1/1)      (-)(1/1)      nd                           Ovary  (-)(2/2)      (-)(1/1)      (-)(2/2)      nd                           Fallopian                                                                            (-)(1/1)      (-)(1/1)      (+ /het)(1)   nd                           Tube                                                                          Trachea                                                                              nd            (++++ epith.)(1/1)                                                                          nd            nd                           Placenta                                                                             nd            (++++ fetal endoth.)(1/1)                                                                   nd            nd                           NORMAL CYNOMOLGUS MONKEY TISSUES                                              Liver  (-)(1/1)      (-)(1/1)      (-)(1/1)      (-)(1/1)                     Kidney (-)(occ. glom.)(1/1)                                                                        (+ ap dt, gl. caps.)(1/1)                                                                   (+ ap tub, gl. caps.)(1/1)                                                                  (-)(1/1)                     Brain  (-)(2/2)      (-)(2/2)      (-)(1/1)      (-)(2/2)                     Cerebellum                                                                           nd            nd            (-)(1/1)      nd                           Spinal Cord                                                                          (-)(1/1)      (-)(1/1)      (-)(2/2)      (-)(1/1)                     Periph.                                                                              nd            nd            (-)(1/1)      nd                           Nerve                                                                         Spleen nd            nd            (-)(1/1)      nd                           Lymph Node                                                                           nd            nd            (-)(1/1)      nd                           Skin   (-)(1/1)      (-)(1/1)      (-, ex. + seb. glnds)(1/1)                                                                  nd                           Skeletal                                                                             nd            nd            (-)(1/1)      nd                           Muscle                                                                        Esophagus                                                                            (+++ diff epith)(1/1)                                                                       (+++ diff epith)(1/1)                                                                       (+++ diff epith)(1/1)                                                                       (+++ diff epith)(1/1)        Small Bowel                                                                          (-)(1/2)(+ muc gran)(1/2)                                                                   (-)(2/2)      (++ ap muc. B glnds)(1/1)                                                                   (-)(2/2)                     Stomach                                                                              (het + glnds)(1/2)                                                                          (+++ glnds)(2/2)                                                                            (+++ glnds)(1/1)                                                                            (+++ glnds, het.)(1/1)       Colon  (-)(1/1)      (-)(1/1)      nd            (-)(1/1)                     Bladder                                                                              (-)1/1)       (+/-)(1/1)    (+++ epith)(1/1)                                                                            nd                           Pancreas                                                                             (-; + muc)(2/2)                                                                             (-; + muc)(3/3)                                                                             (+++ het ac.)(2/2)                                                                          (+/- het)(2/2)               Salivary                                                                             (het +++ a & d)(2/2)                                                                        (++ het a; ++ d)(2/2)                                                                       (++ het a; ++ d)(3/3)                                                                       (++ het a; ++ d)(2/2)        Gland                                                                         Mammary                                                                              (het +)(1/1)  (het. + g; ++ d)(1/1)                                                                       (het. ++ g & d)(1/1)                                                                        nd                           Glands                                                                        Thyroid                                                                              (-)(1/1)      (-)(1/1)      (-)(3/3)      (-)(1/1)                     Parathyroid                                                                          nd            nd            (-)(1/2)(het +)(1/1)                                                                        nd                           Ovary  nd            nd            (-)(1/1)      nd                           Vaginal                                                                              nd            nd            (++++)(1/1)   nd                           Glands                                                                        Uterine                                                                              (-)(1/1)      (-)(1/1)      (++ apic. muc.)(1/1)                                                                        nd                           Cervix                                                                        Uterine                                                                              (-)(1/1)      (+ deep glands)(1/1)                                                                        (++ deep glands)(1/1)                                                                       nd                           Endometrium                                                                   Thymus (-)(1/1)      (-)(1/1)      (-)(1/1)      nd                           Trachea                                                                              (-)(1/1)      (+ apic epith, het gl.)(1/1)                                                                (+ apic epith & gl)(1/1)                                                                    nd                           Tongue (-)(1/1)      (+ diff epith)(1/1)                                                                         (++ diff epith)(1/1)                                                                        nd                           __________________________________________________________________________     Immunohistochemical analysis was performed on cryostat sections of            freshfrozen tissues, postfixed in acetone and incubated with primary          antibodies at 10 μg/ml except where indicated. Labeling was then           performed using affinitypurified goat antimouse IgG conjugated to             horseradish peroxidase, developed using diaminobenzidine, then treated        with hematoxylin followed by osmium tetroxide.                                (- = no localization;                                                         + = moderate;                                                                 ++ strong)                                                                    (x/y = x examples of this pattern seen in y samples tested)                   (het = heterogeneous)                                                         nd = not determined.                                                     

As shown in Table I, the B1, B3 and B5 reactive epitopes are all foundin varying amounts in the mucins of the stomach and small bowel, in thedifferentiated cell layer in the esophagus, in the epithelia of thetonsil, trachea and urinary bladder. These epitopes or antigens, whichreact with the B1, B3 and B5 monoclonal antibodies, can also be found invarious other epithelia in a heterogeneous distribution, such as in thepancreas, salivary gland and mammary gland. B3 has the ability to reactwith the fetal endothelium, suggesting that the B3 monoclonal antibodyrepresents an antigen expressed in fetal development.

Furthermore, as shown in Table I, the overall pattern of reactivity ofthe B1, B3 and B5 antibodies is different from the pattern of reactivityof a previously isolated antibody termed BR96 (Hellstrom et al., CancerRes. 50:2183-90 (1990)). BR96 demonstrates some of the same reactivitypatterns as observed with B1, B3 and B5. For example, BR96 reactivity isparticularly notable in distal tubules in human kidney, as is B1 and B3reactivity. However, there are distinct differences between these fourantibodies in certain sites, such as in kidneys tubules, type IIpneumocytes in the lung, mucin in the colon and in pancreatic ducts. Forexample, BR96 reactivity is not present in mucin in normal human colonsample, as is B5 reactivity. Such information can be significant indetermining whether a monoclonal antibody administered for therapeuticpurposes will be toxic with respect to normal tissues.

The above four antibodies can also be evaluated in normal monkey tissues(See Table I). Tissues similar to those in the human samples arereactive; however, similar to the human tissues, there are distinctdifferences between B1, B3, B5 and BR96 reactivity in kidney tubules,small bowel mucin, bladder epithelium, pancreas, cervical mucin,endometrial glands, and the epithelium of trachea and tongue. Thesedifferences indicate that each of these antibodies recognize differentepitopes. Thus, the chemical structures of the epitopes are different.

Various cancer cell lines can also be examined for reactivity with B1,B3, B5 and BR96 using immunofluorescence. The results of such a studyare shown in Table II presented below.

                  TABLE II                                                        ______________________________________                                        Immunofluorescence Localization of B1, B3, B5 and BR96                        on Human Cultured Cell Lines                                                  CELL LINE    B1       B3       B5     BR96                                    ______________________________________                                        A431 (epidermoid Ca)                                                                       +++      ++++     ++++   ++++                                                 het      het      het                                            MCF-7 (breast Ca)                                                                          ++++     ++++     ++++   ++++                                    OVCAR-3      -        -        ++++   ++++                                    (overian Ca)                   het                                            KB (cervical Ca)                                                                           -        +/-      ++++   -                                                             het      het                                            HT-29 (colon Ca)                                                                           +        ++++     ++++   +/-                                                                    het                                            MDA-MB-468   ++++     ++++     nd     ++++                                    (breast Ca)                                                                   DU145 (prostate Ca)                                                                        +        ++       ++++   ++++                                                 het      het      het                                            HTB20 (breast Ca)                                                                          +++      +++      ++++   ++++                                                                   het                                            HTB33 (cervical Ca)                                                                        +++      +++      ++++   ++++                                                          het      het                                            ______________________________________                                         Het = heterogeneous;                                                          (-) = negative;                                                               (+ = weakly positive;                                                         ++ = moderate;                                                                +++ = strong;                                                                 ++++ = very strong).                                                          ng = not determined.                                                     

As clearly shown in Table II, B1, B3, B5 and BR96 react with some celllines uniformly. However, there are differences in reactivity,especially for OVCAR-3, KB and HT-29 cells. Again such data suggeststhat the epitopes recognized by four antibodies are different from astructural standpoint. Furthermore, such differences in epitopestructure and therefore in reactivity with monoclonals may be anadvantage in therapy in some patients.

Tumors can also be examined for the expression of antigens which reactwith the 4 antibodies, using peroxidase immunohistochemistry. Table III(below) shows that the B1, B3, B5 and BR96 antibodies react well withcarcinomas of colon and gastric origin, and mucinous ovarian carcinomas.Reactivity can be detected in a smaller number of breast, esophageal andother carcinomas.

                                      TABLE III                                   __________________________________________________________________________    IMMUNOHISTOCHEMICAL REACTIVITIES OF HUMAN TUMORS WITH B1, B3, B5 AND          BR96                                                                                  B1             B3            B5           BR96                        __________________________________________________________________________    Colon Ca                                                                              (++ het cells & mucin)(3/3)                                                                  (++++ cells & mucin)                                                                        (+++ het)(3/3)                                                                             (+++)(3/3)                                         (9/12)(het ++)(3/12)                                   Gastric Ca                                                                            (++++ cells & mucin)(3/4)                                                                    (++++)(3/4)   (+++)(1/1)   (++++)(3/4)                 Ovarian Ca                                                                            (mucinous = +++ cells &                                                                      (mucinous = ++++)(2/2)                                                                      (mucinous = +++)(1/1)                                                                      (+++ mucinous)(1/1)                 mucin)(1/1); (cystadencoCa =                                                                 (cystadenoCa:(+++)(4/20)                                                                    (cystadenoCa:het +++)                                                                      (+ het cystadenoCa)                 het +)(2/3)    (het ++)(6/20)                                                                              (2/2)        (1/2)                       Breast Ca                                                                             (het +)(1/2)   (+++ het)(14/21)                                                                            (-)(2/2)     (+++)(5/7)                  Esophageal Ca                                                                         nd             (het +)(4/9); (++++)                                                                        nd           nd                                                 (3/9)                                                  Prostate                                                                              nd             nd            (+++)(1/1)   (++ het)(2/2)               Cervical Ca                                                                           nd             (het +)(1/1)  (-)(1/1)     nd                          Endometrial Ca                                                                        nd             (het ++)(1/1) (++)(1/1)    nd                          Lung Ca nd             (het +)(1/3)  nd           nd                          __________________________________________________________________________     Immunohistochemical analysis was performed on cryostat sections of            freshfrozen tissues, postfixed in acetone and incubated with primary          antibodies at 10 μg/ml except where indicated. Labeling was then           performed using affinitypurified goat antimouse IgG conjugated to             horseradish peroxidase, developed using diaminobenzidine, then treated        with hematoxylin followed by osmium tetroxide.                                (- = no localization;                                                         + = moderate;                                                                 ++ = strong)                                                                  (x/y = x examples of this pattern seen in y samples tested)                   (het = heterogeneous)                                                         nd = not determined.                                                     

The results of Tables I, II and III indicate that B1, B3 and B5 reactwith many common tumors and appear to react with a limited number ofnormal tissue sites. In addition, these antibodies show distinctdifferences in reactivity in some varying tissue samples indicating thatthe precise epitopes they detect are different.

When MCF-7 cells bearing the B1, B3 and B5 epitopes are metabolicallylabelled using radioactive amino acids, then extracted and the extractsimmunoprecipitated, the reactive species of molecules that areprecipitated by B1, B3 and B5 can be analyzed by gel electrophoresis andauto radiography. B1 and B3 specifically immunoprecipitate protein bandsof a very high molecular weight (>250,000 Daltons), consistent withtheir reactivity with high molecular weight mucins. Because B5 is an IgMantibody, for technical reasons this method is unable to showspecifically reactive proteins.

Monoclonal antibodies B1, B3 and B5 can serve as targeting agents forthe construction of immunotoxins, in which the monoclonal antibody islinked to a toxin, for example, Pseudomonas exotoxin (see Table IVbelow). As previously disclosed in Pastan, et al. (U.S. Pat. No.4,545,985) conjugates of Pseudomonas exotoxin and monoclonal antibodiesshow efficacy in killing cells that are targeted by the epitope-reactivesite of the antibodies. Constructions made by linking B1, B3 or B5 tosuch toxin would then be introduced into patients that had tumors thatwere reactive with these monoclonal antibodies, and the immunotoxinswould bind to and kill the tumor cells within the patient. Normaltissues that were also reactive with these monoclonal antibodies wouldbe affected if the antigenic sites were accessible to the bloodcirculation. In the case of many of the sites of expression of antigensreactive with B1, B3, and B5, the reactive epitope appears byimmunohistochemistry to be relatively inaccessible to the circulation,such as the reaction with mucins in the lumen of the gastrointestinaltract. Thus, it is not possible to predict with total certainty what thetoxic effects of such immunotoxins would be in a human patient. Tumorcells that express these surface antigens, however, are rarely in alocation that would render them inaccessible to the immunotoxin, and thetumor cells should therefore, be susceptible to targeted cell killing byimmunotoxins constructed with B1, B3 or B5.

                  TABLE IV                                                        ______________________________________                                        Activity of Immunotoxin Composed of B3 and a                                  Pseudomonas Exotoxin Mutant in Which Lysine 57                                is Converted to Arginine (B3-PE.sup.Arg57).                                                 ID.sub.50                                                                       B3-PE.sup.Arg57                                                                         MOPC-PE.sup.Arg57                                   Cell Line       ng/ml     ng/ml                                               ______________________________________                                        A431 Epidermoid Ca                                                                            0.2       >100                                                MCF-7 Breast Ca 0.3       >100                                                ______________________________________                                         ID.sub.50 is the concentration of agent that inhibits protein synthesis b     50% in a 16 hour incubation.                                             

In addition to bacterial or plant toxins conjugated to monoclonalantibodies, other effector agents may be used together with targetedmonoclonal antibodies to treat or diagnose human cancer. For example,radionuclides conjugated to antibodies that bind to tumors can producecell killing based on the high local concentration of radiation.Chemotherapeutic drugs, for example, vinblastine or daunomycin, can becoupled to antibodies and delivered at high concentration to cells thatreact with the antibodies. B1, B3, and B5 may provide a targetingmechanism for such combination of effector agents that could producesuccessful regression of reactive human tumors when introduced intopatients.

In addition to the targeting of immunotoxins to tumors in a cancerpatient, these antibodies also recognize materials such as surfacemucins on tumor cells that would be expected to be shed into thesurrounding tissues, picked up by the blood stream, and detectable inblood samples taken from distant sites. Such shed antigens have provento be useful in the diagnosis of primary and recurrent cancers usingantibodies that react to these shed antigens. A currently useful exampleof this is the CA125 antigen that can be assayed in sera from patientswith ovarian cancer to predict recurrence or to confirm primarydiagnosis of tumor. It is possible, therefore, that B1, B3 and B5 may beuseful in the diagnosis of tumors.

Also, the selective reactivity of these antibodies with certain types oftumor cells could be exploited for anatomic pathological diagnosis oftumors, clarifying the type and origin of tumors, and whether aparticular group of cells represents a recurrence of a previous tumor orthe development of another primary tumor elsewhere. Such a diagnosticdetermination can be useful for the subsequent planning of anti-tumortherapy in each particular patient. In particular, immunohistochemicalpathologic diagnosis in tissue sections (e.g., biopsies) or cytologicalpreparations (e.g., Pap smears, effusions) can be performed using themonoclonal antibodies of the present invention.

Another potential use of such targeting antibodies could be in thediagnosis of macroscopic foci of tumor using antibodies B1, B3 or B5coupled to radioisotopes that could be detected either by external bodyscanning (imaging diagnosis) or by localization using radiation detectorprobes at the time of exploratory surgery.

In addition to the initial clones of B1, B3 and B5 isolated as mousemonoclonal antibodies, variations of the constant regions of theseantibodies incorporating constant regions of other species, such ashuman, could be performed, in which the resulting antibody would displayless immunogenicity as a foreign antigen itself when introduced into ahuman patient. Pharmaceutical compositions can also be made using themonoclonal antibodies.

Also, the genes responsible for the variable regions of these antibodiescould be isolated and targeting agents constructed using these variableregion genes in tandem with genes for other proteins, such as toxingenes, or other effector proteins that could direct cell killing eitherdirectly or through the activation of endogenous mechanisms, such as theimmune system. The variable regions of immunoglobulin genes encode theantigen binding site which enables the chimeric antibody toxin proteinto bind to and kill target cells expressing the antigen reacting withantibodies B1, B3, and B5.

The present invention can be illustrated by the use of the followingnon-limiting examples.

EXAMPLE I

Production of the B1, B3 and B5 Monoclonal Antibodies.

The human tumor cell lines OVCAR-3, KB, MCF-7, HT-29, MDA-MD-468, DU145,HTB20, and HTB33 have been previously described (Hay et al., AmericanType Culture Collection Catalog of Cell Lines and Hybridomes, 6th Ed.(1988)). For antibodies B1 and B3, mice were tolerized to normal humankidney membranes (Matthew et al., J. Immunol. Methods 100:73-82 (1978)and immunized with MCF-7 cells using methods previously described(Willingham et al., Proc. Natl. Acad. Sci. USA 84:2474-78 (1987)). Forantibody B5, mice were not tolerized and were immunized with A431 cells.Spleens from immunized mice were removed and the suspended cells werefused with AG8 mouse myeloma cells. The resulting clones were screenedtwo weeks later employing the ScreenFast (Life Technologies, Inc.Gaithersburg, MD) large scale screening chamber using rhodamine indirectimmunofluorescence on living MCF-7 and A431 cells for B1 or B3, and B5,respectively. Selected clones were secondarily screened using peroxidaseimmunohistochemistry on cryostat sections of human tumors and mammaliantissues. Clones B1, B3 and B5 were selected that reacted with humancolon and gastric cancers, and not with normal human liver, kidney orcolon. After sub-cloning, the isotypes of these clones was determined tobe IgG_(1k) for clones B1 and B3, and IgM for clone B5. Antibody waspurified from the supernatants of these clones using serum-free definedculture media and ammonium sulfate precipitation. A hybridoma cell linesecreting antibody B1 has been deposited with the American Type CultureCollection ("ATCC"), in Rockville, Md., on Oct. 12, 1990 and designatedAccession No. HB10572. A hybridoma secreting antibody B3 was depositedwith the ATCC on Oct. 12, 1990 and designated Accession HB10573. Ahybridoma secreting antibody B5 was deposited with the ATCC on Oct. 10,1990 and designated Accession No. HB10569.

EXAMPLE II

Determination of Distribution of Antigens Reactive with Antibodies B1,B3 and B5 In Human Tumor-Free Tissues,

Human Tumors And Monkey Tissues.

Samples of normal human tissues, cynomolgus monkey tissues, and humantumors were fresh-frozen and cryostat sections were prepared forperoxidase immunohistochemistry as previously described (Willingham,FOCUS 12:62-67 1990)) using B1, B3 and B5 as primary antibodies.Localization of antibodies was detected by development of the peroxidasesubstrate reaction using diaminobenzidines. Tissues sectionsdemonstrated major reactives of B1, B3 and B5 in the epithelium of thetonsil, stomach, esophagus, and bladder, as well as in mucins of thesmall bowel and colon. Similar localization was found in monkey tissuesin esophagus, small bowel, stomach, bladder, salivary gland, andpancreas with some differences being noted between the differentantibodies (see Table I). Human tumors showed strong reactivity for B1,B3 and B5 in carcinomas of colon, stomach, ovary, and esophagus, withvariable localization seen in carcinomas from breast, cervix, prostate,endometrium and lung (See Table III). All localizations, except as notedin Table I, represented antigen reaction that appeared to be on thesurface of the cells, making these sites potential targets forimmunotherapy.

EXAMPLE III

Determination of the Effectiveness of BE-PE As An Anti-Tumor Agent

B3 was coupled to Pseudomonas exotoxin as previously described(Willingham et al., Proc. Natl. Acad. Sci. USA 84:2474-78 (1987)). To dothis, a mutant form of PE in which lysine 57 of PE was mutated toarginine (PE^(Arg) 57) was used. The immunotoxin was purified and testedin tissue culture where it was shown to kill target A431 and MCF-7 cells(Table V). A control antibody (MOPC 21) was also coupled to PE^(Arg) 57and it had no cell killing activity. B3-PE^(Arg) 57 was then givenintraperitoneally to mice. The mice had been implanted with 3 millionA431 cancer cells on day 0, and by day 4 had small cancers which wererapidly growing. The immunotoxin was given IP on days 4, 6, and 8 and,as shown in FIG. 1, the tumors regressed and apparently disappeared,whereas, in the control animals treated with diluent, the tumors grewrapidly.

What is claimed is:
 1. A method of diagnosing a tumor bearing an antigenreactive with antibody B1 secreted by the hybridoma cell line bearingATCC No. HB10572 in a patient comprising: administering to the patient aradioisotope coupled to an antibody having the tissue bindingspecificity of antibody B 1 and detecting for the presence of the tumorby imaging using radiation detection methods.
 2. A method of diagnosinga tumor bearing an antigen reactive with antibody B3 secreted by thehybridoma cell line bearing ATCC Designation No. HB10573 in a patientcomprising: administering to the patient a radioisotope coupled to anantibody having the tissue binding specificity of antibody B3 anddetecting for the presence of the tumor by imaging using radiationdetection methods.
 3. A method of diagnosing a tumor bearing an antigenreactive with antibody B5 secreted by the hybridoma cell line bearingATCC Designation No. HB10569 in a patient comprising: administering tothe patient a radioisotope coupled to an antibody having the tissuebinding specificity of antibody B5 and detecting for the presence of thetumor by imaging using radiation detection methods.